Influenza viruses have been a major cause of mortality and morbidity in man throughout recorded history. Epidemics occur at regular intervals which vary widely in severity but which always cause significant mortality and morbidity, most frequently in the elderly population. The cause of influenza epidemics was first attributed to a virus by R. E. Shops, who showed that influenza epidemics could be transmitted with filtered mucus. Influenza viruses are currently divided into three types: A, B, and C, based upon differences in internal antigenic proteins.
An influenza infection produces an acute set of symptoms including headache, cough, fever and general malaise. In severe cases or situations involving pre-existing pulmonary or cardiovascular disease, hospitalization is required. Pneumonia due to direct viral infection or due to secondary bacterial or viral invasion is the most frequent complication. For a review on the clinical aspects of influenza virus infection see Douglas, R. G., New England Journal of Medicine, 322:443-450 (1990).
New strains of influenza caused by antigenic drift appear at regular frequency, usually annually, and begin a cycle of infection which travels around the globe. Little is known about how individual epidemics are initiated. Major new subtypes of influenza appear less frequently but can result in major pandemics.
The most effective way to deal with the influenza virus for the population at risk of severe complications is by prevention. Use of the available influenza vaccine is an effective way to lower the mortality in a population, however due to the ever-changing nature of the virus, the development of a vaccine with the appropriate composition to protect against the currently circulating virus strains is complex and expensive. Moreover, patient compliance in receiving the vaccine is generally very low. Thus large numbers of patients at risk of serious complications from influenza virus go unprotected.
There are several drugs available which have some activity against the influenza virus prophylactically. None, however, are effective against influenza type B. Moreover, they are generally of very limited use therapeutically, and have not been widely used in treating the disease after the onset of symptoms. Accordingly, there is a world-wide need for improved therapeutic agents for the treatment of influenza virus infections.
Prior attempts at the inhibition of influenza virus using antisense oligonucleotides have been reported. Leiter and co-workers have targeted phosphodiester and phosphorothioate oligonucleotides to influenza A and influenza C viruses. Leiter, J., Agrawal, S., Palese, P. & Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 87:3430-3434(1990). These workers targeted only the polymerase PB1 gene and mRNA in the vRNA 3' region and mRNA 5' region, respectively. Sequence-specific inhibition of influenza A was not observed although some specific inhibition of influenza C was noted. No other influenza virus segments or mRNA's were targeted.
Zerial and co-workers have reported inhibition of influenza A virus by oligonucleotides coincidentally linked to an intercalating agent. Zerial, A., Thuong, N. T. & Helene, C., Nucleic Acids Res., 57:9909-9919 (1987). Zerial et al. targeted the 3' terminal sequence of 8 vRNA segments. Their oligonucleotide analog was reported to inhibit the cytopathic effects of the virus in cell culture. EP Patent 169787, Helene et al. disclose oligonucleotide compounds covalently bound to an intercalating group and complementary with a nucleic acid sequence involved in replication of a nucleic acid and of transcription and/or translation of one or more genes; oligonucleotides covalently bound to an intercalating group and complementary with a sequence for replicating or developing a virus or bacterium or parasite; and oligonucleotides covalently bound to an intercalating group and complementary with a sequence for replicating or developing the influenza or herpes virus, or with an oncogene.
European Patent Application No. 82110494.0 (Krug et al.) discloses oligonucleotides containing a 5' methylated cap structure to increase the affinity of the oligonucleotide for influenza viral endonuclease and transcriptase. In addition, capped oligonucleotides are modified to prevent them from acting as primers, e.g., being less than 10 nucleotides in length; or extended to contain a 3' terminal deoxymononucleotide or a 3' terminal 3'-O-methylated ribonucleotide; or having at least 14 nucleotides modified in the sugar and/or base moieties and/or in the nucleotide bond.
Kabanov and co-workers have synthesized an oligonucleotide complementary to the loop-forming site of RNA encoding RNA polymerase 3. Kabanov, A. V., Vinogradov, S. V., Ovcharenko, A. V., Krivonos, A. V., Melik-Nubarov, N. S., Kiselev, V. I. & Severin, E. S., FEBS, 259:327-330 (1990). Their oligonucleotide was conjugated to a undecyl residue at the 5' terminal phosphate group. They found that their oligonucleotide inhibited influenza A virus infection in MDCK cells.
Although each of the foregoing workers reported some degree of success in inhibiting some function of an influenza virus, a general therapeutic scheme to target influenza viruses has not been found. Moreover, improved efficacy is required in influenza virus therapeutics. Accordingly, there has been and continues to be a long-felt need for the design of antisense oligonucleotide analogs which are capable of effective therapeutic use. This long-felt need has not been satisfied by prior work in the field of antisense oligonucleotide therapy for influenza. Others have failed to identify target sites in which antisense oligonucleotides or oligonucleotide analogs are therapeutically effective at reasonable rates of application.